The present invention relates to methods and devices for analyzing nucleic acid samples for sequence variations at multiple loci. More particularly, the invention relates to the use of differential fluorescent emission and differential hybridization melting temperature of fluorescently labeled nucleotide probes to identify nucleic acid samples, including PC and LCR products, which have multiple potential sequences at multiple loci. The invention also relates to devices which allow simultaneous analysis of three or more fluorescent labels having different emission spectra.
The continued discovery of novel genes provides a resource of genetic material for studying the association between genotype and disease. As databases for polymorphic markers and disease causing mutations continue to grow, there is an increasing need for procedures that can screen nucleic acid sequences for the presence of known polymorphisms and mutations. Optimally, the procedure should be capable of analyzing multiple DNA sites simultaneously (including nucleic acid loci that are physically separated by great distances) for the presence of mutations or polymorphisms.
Current methods for determining the genetic constitution of individuals (genotyping) include oligonucleotide ligation, allele-specific oligonucleotide hybridization, and PCR-restriction fragment length analysis. All these methods require time consuming multiple manual steps. One alternative method of genotyping uses the melting temperature of fluorescent hybridization probes that hybridize to a PCR-amplified targeted region of genome/nucleic acid sequence to identify mutations and polymorphisms.
The polymerase chain reaction (PCR) is a technique of synthesizing large quantities of a preselected DNA segment. The technique is fundamental to molecular biology and is the first practical molecular technique for the clinical laboratory. PCR is achieved by separating the DNA into its two complementary strands, binding a primer to each single strand at the end of the given DNA segment where synthesis will start, and adding a DNA polymerase to synthesize the complementary strand on each single strand having a primer bound thereto. The process is repeated until a sufficient number of copies of the selected DNA segment have been synthesized. During a typical PCR reaction, double stranded DNA is separated into its single strands by raising the temperature of the DNA containing sample to a denaturing temperature where the two DNA strands separate (i.e., the xe2x80x9cmelting temperature of the DNAxe2x80x9d) and then the sample is cooled to a lower temperature that allows the specific primers to attach (anneal), and replication to occur (extend). Currently preferred methods utilize a thermostable polymerase in the polymerase chain reaction. A preferred thermostable DNA polymerase for use in the PCR reaction is the Taq DNA Polymerase and derivatives thereof, including the Stoffel fragment of Taq DNA polymerase and KlenTaql polymerase (a 5xe2x80x2-exonuclease 1 deficient variant of Taq polymerase (see U.S. Pat. No. 5,436,149).
Other nucleic acid amplification procedures are also widely practiced. For example, the self-sustained sequence replication (3SR) reaction utilizes three enzymes. The 3SR method is described in Guatelli et al., PNAS (USA) 87:1874-1878 (1990) and Mueller et al., Histochem. Cell Biol. 108(4-5):431-437 (1997). A similar method is described in U.S. Pat. No. 5,399,491. Strand displacement amplification (SDA) is another method of isothermal nucleic acid amplification. SDA relies on primer-directed nicking activity of a restriction enzyme and strand replacement activity of a polymerase which is exonuclease-deficient. SDA is described in Walker et al., PNAS (USA) 89:392-396 (1992); Walker et al., Nucleic Acids Res. 20(7):1691-1696 (1992); Nadeau et al., Anal. Biochem. 276(2):177-187 (1999) and in U.S. Pat. Nos. 5,270,184, 5,422,252, 5,455,166, and 5,470,723. Yet another method, rolling-circle amplification (RCA) utilizes DNA polymerase to replicate circularized oligonucleotides. RCA is described in Lizardi et al., Nat. Genet. 19(3):225-232 (1998) and U.S. Pat. No. 5,854,033. Each of the above-cited references is incorporated herein in its entirety.
Thermal cycling may be carried out using standard techniques known to those skilled in the art, including the use of rapid cycling PCR. Rapid cycling techniques are made possible by the use of high surface area-to-volume sample containers having relatively high thermal conductivity. The use of high surface area-to-volume sample containers allows for a rapid temperature response and temperature homogeneity throughout the biological sample. Improved temperature homogeneity also increases the precision of any analytical technique used to monitor PCR during amplification.
In a method compatible with the present invention, amplification of a nucleic acid sequence is conducted by thermal cycling the nucleic acid sequence in the presence of a thermostable DNA polymerase. The method comprises the steps of placing a biological sample comprising the nucleic acid sequence in a PCR vessel, raising the temperature of the biological sample from a first temperature to a second temperature, wherein the second temperature is at least 15xc2x0 C. higher than the first temperature, holding the biological sample at the second temperature for a predetermined amount of time, lowering the temperature of the biological sample from the second temperature to at least the first temperature and holding the biological sample at a temperature at least as low as the first temperature for a pre-determined length of time. The temperature of the biological sample is then raised back to the second temperature, and thermal cycling of the biological sample is repeated a predetermined number of times. In one embodiment, the method of amplifying a DNA sequence comprises a two temperature cycle wherein the samples are cycled through a denaturation temperature and an annealing temperature for a predetermined number of repetitions. However the PCR reaction can also be conducted using a three temperature cycle wherein the samples are cycled through a denaturation temperature, an annealing temperature and an elongation temperature for a predetermined number of repetitions.
In the state of the art for PCR, each temperature cycle of the PCR reaction is completed in approximately 60 seconds or less. Rapid cycling times can be achieved using the device and techniques described in U.S. Pat. No. 5,455,175, the disclosure of which is expressly incorporated herein.
PCR amplification of one or more targeted regions of a DNA sample has been conducted in the presence of fluorescently labeled hybridization probes, wherein the probes are synthesized to hybridize to a specific locus present in a target amplified region of the DNA. Many different probes are available for monitoring PCR each cycle. Dyes like ethidium bromide or SYBR Green I, which preferentially bind to double-stranded DNA, can be used in any amplification and a re inexpensive. Although not sequence specific, product specificity can be increased by analysis of melting curves (Ririe et al., Anal. Biochem. 245:154-160 (1997)), or by acquiring fluorescence at a high temperature where nonspecific products have melted (Morrison et al., BioTechniques 24(6):954-958, 960, 962 (1998)). However, multiplexing by color is not possible.
Multiplexing by color is possible with dual-labeled oligonucleotides, including hairpin primers (Sunrise(trademark) primers), hairpin probes (Molecular Beacons(trademark)), and exonuclease probes (TaqMan(trademark) probes). Hairpin primers include one fluorophore and one quencher (Nazarenko et al., Nucl. Acids Res. 25:2516-2521 (1997)). Hairpin probes hybridize internal to the primers and are sequence specific (Tyagi et al., Nature Biotechnology 14:303-308 (1996)). Exonuclease probes are cleaved during polymerase extension by 5xe2x80x2-exonuclease activity (Livak et al., PCR Meth. Appl. 4:357-362 (1995)). All these dual-labeled probes require careful design and are expensive. Their synthesis is difficult, requiring manual addition of at least one label and high pressure liquid chromatography for purification.
An alternative sequence specific method has been developed wherein two oligonucleotide probes that hybridize to adjacent regions of a DNA sequence are used (Wittwer et al., BioTechniques 22:130-138 (1997)). Each oligonucleotide probe is labeled with a respective member of a fluorescent energy transfer pair. In this method, the presence of the target nucleic acid sequence in a biological sample is detected by measuring fluorescent energy transfer between the fluorophores on the two labeled oligonucleotides. Such an energy transfer event is indicative of the presence of the target nucleic acid sequence.
The present invention provides methods and devices for analyzing sequence variations in nucleic acid samples using multiple colors. In one aspect, a method for analyzing a nucleic acid comprising multiple loci, each having two, three or more possible allelic sequences. The method involves combining at least a first and second pair of oligonucleotide probes with the nucleic acid sample. The first pair of probes is capable of hybridizing in proximity to each other within a segment of the nucleic acid sample comprising the first locus and the second pair is capable of hybridizing in proximity to each other within a segment of the nucleic acid sample comprising the second locus. The first member of each probe pair comprises a FRET donor and the second member comprises a FRET acceptor, the FRET acceptor of the first probe pair member having a different emission spectrum from the FRET acceptor of the second probe pair. Upon hybridization, the proximity of the first and second member of each probe pair is sufficient to allow fluorescence resonance energy transfer between the FRET donor and the FRET acceptor. At least one of the members of the first probe pair has a sequence which results in the differential hybridization of that member with at least two different alleles which may be present at the first locus and at least one of the members of said second pair has a sequence which results in the differential hybridization of that member with at least three different alleles which may be present at the second locus. The method further involves measuring the emission of each of the FRET acceptors at a first temperature and repeating those emission measurements at a second and third temperature. The emission of the FRET acceptors at different temperatures provides an indication of the alleles present at the first and second loci. In a preferred embodiment, the method further involves a third pair of oligonucleotide probes which is combined with the nucleic acid sample, wherein the FRET acceptor of each of the first, second and third probe pairs has an emission spectrum which is different from the emission spectrum of the others. In a preferred embodiment, the nucleic acid sample is the product of one or more reactions selected from the group consisting of PCR, 3SR, SDA and RCA. In a preferred embodiment, at least one probe pair member has two FRET acceptors, two FRET donors or a FRET acceptor and a FRET donor, and is a member of two different probe pairs.
In another aspect, the invention provides a method for analyzing a nucleic acid sample comprising three or more loci each having at least two different allelic sequences. This method involves combining at least a first, a second and a third pair of oligonucleotide probes with the nucleic acid sample, each of the members of the pairs being capable of hybridizing in proximity to each other within a segment of the nucleic acid sample comprising at least one of the multiple loci. The first member of each pair comprises a FRET donor and the second member comprises a FRET acceptor, wherein the FRET acceptor in the first pair has an emission spectrum which is different from the emission spectrum of the FRET acceptor in the second and third oligonucleotide probe pairs. When the second and third probe pairs have the same FRET acceptor, each of the second and third probe pairs has a different Tm from each other for each different allele within the nucleic acid sample segment to which each member hybridizes. Upon hybridization, the proximity of the probe pair members is sufficient to allow fluorescence resonance energy transfer between the FRET donor and the FRET acceptor. At least one of the members of each pair has a sequence which results in the differential hybridization of that member with at least two different alleles which may be present at said loci. The method further involves measuring the emission of each of the FRET acceptors at a first temperature and repeating those emission measurements at a second and third temperature. The emission of the FRET acceptors at different temperatures provides an indication of the alleles present at the multiple loci. In a preferred embodiment, the FRET acceptor of each of the second members of each of a first, a second and a third probe pair has an emission spectrum which is different from the emission spectrum of the others. In a preferred embodiment, the nucleic acid sample is the product of one or more reactions selected from the group consisting of PCR, 3SR, SDA and RCA. In a preferred embodiment, at least one probe pair member has two FRET acceptors, two FRET donors or a FRET acceptor and a FRET donor, and is a member of two different probe pairs.
In addition, provided herein is a method for analyzing a nucleic acid sample. The method involves contacting a nucleic acid sample comprising multiple loci with at least a first and a second primer, each of which is specific for one of said loci, under conditions which allow formation of at least a first and a second linear amplification product which are specific for each of said loci. The first amplification product contains one of at least two or more different allelic sequences which may be present at each of the loci within the first amplification product and the second amplification product contains one of at least three or more different allelic sequences which may be present at each of the loci within the second amplification product. Each of the amplification products comprises at least one member of a pair of FRET acceptor or FRET donor. The method further involves contacting each of the loci specific amplification products with FRET labeled oligonucleotide probes. Each of the FRET probes hybridizes with the amplification product at a segment encompassing a specific locus, wherein each of the probes has a sequence complementary to one of the allelic sequences which may be present at the specific locus within the amplification products and the hybridization product of each FRET probe with the other allelic sequences which may be present at the specific locus in the amplification products contains one or more mismatches, insertions or deletions which results in differential Tm of the FRET probe from each of the possible allelic sequences within that locus in the amplification products. Each of the FRET probes contains a member of a FRET donor and acceptor pair which is other than the FRET donor or acceptor contained in the corresponding specific amplification product. One of the FRET acceptors has an emission spectrum which is different from the emission spectrum of the other FRET acceptor. The primer sequences and oligonucleotide probe sequences are chosen so that upon hybridization the FRET donor and acceptor for each pair is in close proximity so as to allow fluorescence resonance energy transfer between the FRET donor and said FRET acceptor. The method further involves measuring the emission of each of the FRET acceptors at a first temperature and repeating those emission measurements at a second and third temperature. The emission of the FRET acceptors at different temperatures provides an indication of the alleles present at the multiple loci. In a preferred embodiment, the nucleic acid sample has three or more loci and is contacted with at least three primers under conditions which allow formation of at least three linear amplification products, and each of the FRET acceptors of a first, a second and a third probe donor and acceptor pair has an emission spectrum which is different from the emission spectrum of the others.
Also provided herein is a method for analyzing a nucleic acid sample comprising. This method involves contacting a nucleic acid sample having at least three loci with at least three primers, each of which are specific for one of the loci, under conditions which allow formation of at least three linear amplification products which are specific for each of the loci. Each of the amplification products contains one of at least two different allelic sequences which may be present at each of the loci. Each of the amplification products comprises at least one member of a FRET acceptor and FRET donor pair. The method further involves contacting each of the loci specific amplification products with FRET labeled oligonucleotide probes. Each of the FRET probes hybridizes with the amplification product at a segment encompassing a specific locus, and the hybridization product of each FRET probe with the other allelic sequences which may be present within that locus in the amplification products contains one or more mismatches, insertions or deletions which result in differential Tm of the probe from each of the possible allelic sequences within that locus in the amplification products. Each of the FRET probes contains a member of a FRET donor and acceptor pair which is other than the FRET donor or acceptor contained in the corresponding specific amplification product. One of the FRET acceptors has an emission spectrum which is different from the emission spectrum of the other FRET acceptor. When the FRET donor and acceptor combination of different probes is the same, the Tm of the probe pairs from each different allele within the nucleic acid segment to which each probe hybridizes is different. The primer sequences and oligonucleotide probe sequences are chosen so that upon hybridization the FRET donor and acceptor for each pair is in close proximity so as to allow fluorescence resonance energy transfer between the FRET donor and the FRET acceptor. The method further involves measuring the emission of each of the FRET acceptors at a first temperature and repeating those emission measurements at a second and third temperature. The emission of the FRET acceptors at different temperatures provides an indication of the alleles present at the first and second loci. In a preferred embodiment, each FRET acceptor of the FRET donor and acceptor combination for a first, a second and a third probe has an emission spectrum which is different from the others.
In consideration of each of the methods described above, in a preferred embodiment, measurements of FRET acceptor emission is measured throughout a range of temperatures, preferably from at least 20xc2x0 C. to at most 95xc2x0 C., preferably at least every 0.1 to 10 seconds, preferably while varying the temperature at least 0.01 to 1xc2x0 C. per second. In a preferred embodiment, emission measurements at a particular temperature are simultaneous. In a preferred embodiment, at least one of the FRET acceptors is selected from the group consisting of LC Red 640, Cy 5, Cy 5.5 and LC Red 705. In a preferred embodiment, emission measurements are corrected for spectral overlap between or among the different fluorophores.
In another aspect of the invention, a device is provided for multichannel color analysis of a PCR or LCR reaction. The device comprises a chamber for holding a nucleic acid amplification reaction product comprising an optically transparent wall. The device further comprises a source for providing electromagnetic radiation to said optically transparent wall. In addition, the device has at least four bandpass filters at least two of which are not coplanar. The filters are positioned to simultaneously or sequentially filter fluorescence emissions from the chamber so as to provide filtered multichannel fluorescence signals. Also, the device comprises an optical detector positioned to receive the filtered emission signals. In a preferred embodiment, the bandpass filters which are not coplanar are orthogonal to each other. In a preferred embodiment, the chamber comprises a nucleic acid amplification reaction chamber.
In yet another aspect, the invention provides a device for multichannel color analysis of a nucleic acid amplification reaction. The device comprises a chamber for holding a nucleic acid amplification reaction product comprising an optically transparent wall. The device also comprises a source for providing electromagnetic radiation to said optically transparent wall and at least three dichroic filters and two bandpass filters. The bandpass filters are not coplanar and the dichroic filters are positioned so that the emissions passing through each bandpass filter intersect each others path to simultaneously or sequentially filter florescence emissions from the reaction chamber, thus providing filtered multichannel florescence signals. In addition, the device comprises an optical detector positioned to receive the filtered emission signals. In a preferred embodiment, the chamber comprises a nucleic acid amplification reaction chamber.
Other aspects of the invention will become apparent to the skilled artisan from the following description of the invention.